Chapter 1: Lab-on-a-chip – Reform, Perform, and Transform Free

Their ability to speed up day-to-day laboratory bench-work with reduced time and expenditure makes lab-on-a-chip (LOC) devices an increasingly viable tool in the healthcare domain. These tools have several advantages over conventional approaches, including the ability to manipulate minute volumes of fluid and the integration of numerous laboratory tasks onto a single chip.¹  Microfluidic devices, commonly referred to as LOC devices, are compact platforms that allow for the integration of numerous laboratory tasks onto a single chip and the manipulation of minute volumes of fluids. These tools have a wide range of uses in the drug development process, including disease modelling, drug delivery, and drug screening.^2 ,3  The capacity of LOC devices to speed up and lower the cost of drug discovery along with high throughput and automation capabilities allow for the quicker and more efficient screening of drug-like molecules compared to conventional techniques. As a result, there is a greater probability of discovering potential drug candidates and the drug discovery process can be sped up.⁴  The LOC-based drug delivery system provides several benefits. For instance, drug efficacy can be increased, and side effects can be decreased by accurately delivering medications to designated parts of the body utilizing microfluidic channels. These gadgets can be used to give medications continuously for a long time through controlled release.⁵  Further, LOC tools can be used for disease modelling, enabling researchers to build simulations of disease processes and test prospective treatments’ modality in a sterile setting. This can aid in better understanding of disease mechanisms and detecting possible therapeutic targets. Overall, LOC systems have enormous potential for drug development and, as technology advances, their application is likely to increase.⁶  This chapter aims to provide an overview of the history and timeline of LOC devices, highlighting their evolution from early prototypes to advanced microfluidic systems. The key advancements and breakthroughs that have propelled the development of these devices over the years have been briefed. One of the primary advantages of LOC devices is their ability to miniaturize and automate complex laboratory processes.⁷  With the integration of microfluidics, sensor technologies, and nanomaterials, these devices enable precise control and manipulation of small volumes of samples, reducing the need for bulky equipment and costly reagents. This miniaturization results in faster analysis times, reduced sample and reagent consumption, and improved sensitivity and specificity of tests. LOC devices hold significant potential as point-of-care testing (POCT) diagnostic tools. The ability to perform rapid and accurate analysis at the patient’s bedside or in resource-limited settings has the potential to revolutionize the healthcare system.^8 ,9  These devices can facilitate early disease detection, monitor treatment response, and enable personalized medicine by providing real-time analysis of biomarkers and genetic variations.

Furthermore, the application of LOC devices extends beyond diagnostics. They have found utility in therapeutic designing and discovery processes, facilitating the development of targeted therapies, drug delivery systems, and personalized medicine approaches. The precise control and manipulation of microenvironments provided by LOC devices enable the study of cellular responses to drugs and help optimize therapeutic interventions.¹⁰  The diverse applications and employment of LOC devices in various fields, including biotechnology, environmental monitoring, food safety, and forensics have been widely studied.^9–11  The way these devices have revolutionized sample handling, biochemical analysis, and DNA sequencing, among other areas, has gained attention. However, the adoption of LOC encounters certain challenges such as scalability, standardization, integration, and cost-effectiveness. Addressing these challenges is crucial for their widespread implementation and commercialization.¹¹ 

Looking ahead, the future of LOC devices holds great promise. Advances in nanotechnology, microfabrication techniques, and machine learning (ML) algorithms will likely drive further innovation and enhance the capabilities of these devices. This chapter explores the potential applications and opportunities that lie ahead, including the integration of artificial intelligence (AI) and the internet of things to create smart, integrated LOC systems¹²  along with a brief insight into the market potential of LOC devices. The growing demand for point-of-care (POC) diagnostics, personalized medicine, and rapid testing methods is driving the market for these devices.¹³  The current trends, market size, and growth prospects, highlighting the key players and industry collaborations that are shaping the landscape have been highlighted. Overall, this chapter aims to provide a comprehensive overview of LOC devices, their advantages, potential applications, challenges, and prospects. The transformative impact of LOC devices on various fields and the future developments that will shape healthcare, diagnostics, and biomedical fields have been discussed.

The foundation of LOC technology can be traced back to the utilization of microtechnology fabrication techniques, which enabled the production of the first fluidic chips in the late 1980s. These initial experiments conducted in laboratories served as a source of inspiration for scientists, who established ambitious objectives for the field. These goals remain relevant and influential today.¹⁴  One of the primary aims of LOC technology was to create intricate systems that could consolidate all essential analysis steps onto a single chip, known as a micro total analysis system (μTAS). This vision encompassed the miniaturization of existing laboratory analysis instruments. Consequently, analytical chemists were particularly drawn to the advancement of these microfluidic systems.

During the early 1990s, there was great anticipation surrounding the potential of conducting (bio)chemical analysis anywhere and at any time, such as performing complete blood analysis at a patient’s bedside (POCT).¹⁵  In 1993, Harrison and Manz achieved a significant breakthrough by miniaturizing capillary electrophoresis, which they believed would pave the way for the development of analytical laboratories on a chip.¹⁶  This advancement offered advantages like speed, reduced sample volume requirements, and the possibility of portability.

In the mid-1990s, other scientific communities, including synthetic chemists and biologists, became interested in LOC technology. Researchers were attracted to microscale reactors on chips, as they opened new avenues for research. Biologists, on the other hand, were intrigued by the opportunity to analyze and experiment with living cells (cellomics) on polymer chips.¹⁷  Over the past 15 years, nanotechnology has also made its way into this field, bringing improvements to existing chip components, and introducing novel concepts for separation and detection. In the early 2000s, there was a growing focus on the development of microfluidic platforms for various applications. The integration of multiple functionalities onto a single chip became a key objective.¹⁸  Researchers explored new fabrication techniques and materials to enhance chip performance, reliability, and scalability. In 2002, Quake et al. demonstrated the potential for large-scale integration of microfluidic chips, drawing parallels to electronic integrated circuits. This development held particular significance for pharmaceutical companies. They highlighted the versatility of the fabrication process and valve multiplexing, envisioning applications ranging from high-throughput screening (HTS) to the design of new liquid display technology.¹⁹  Ahn et al. introduced a disposable plastic biochip that incorporates smart passive microfluidics, on-chip power sources, and an integrated biosensor array.²⁰  This breakthrough expanded the possibilities in LOC technology, with the ultimate complexity and applications being limited only by one’s imagination.

Throughout the 2010s, LOC technology made significant strides in various fields. In medicine, there was a notable emphasis on POC diagnostics, enabling rapid and accurate testing at the patient’s bedside or in remote settings. A consortium of Fraunhofer Institutes has developed a modular LOC system called “Fraunhofer ivD-platform” for POC diagnostics, offering on-site analysis at low cost. The system integrates assays, microfluidics, and sensors within a self-contained cartridge, enabling rapid diagnostic testing in 15 minutes.²¹  Another research group, Manage et al. developed a self-contained, disposable, and inexpensive LOC device called cassette polymerase chain reaction (PCR). It enables rapid DNA amplification without the need for pumps or valves. The device uses capillary forces for sample delivery and can accept raw samples like urine and blood, making it suitable for near POCT.²²  These advancements played a crucial role in improving healthcare accessibility, particularly in resource-limited areas. The field also witnessed remarkable progress in single-cell analysis, with the ability to study individual cells and unravel their heterogeneity. This opened new avenues for understanding complex biological processes, disease mechanisms, and personalized medicine. In 2015, a LOC device developed by Mahshid et al. utilized micro/nano-fabricated features and convex lens-induced confinement (CLIC) for analyzing single cells in real-time. It enables trapping, lysis, genomic DNA extraction, and stretching of long DNA molecules for efficient single-cell analysis with optical interrogation.²³  LOC platforms integrated high-resolution imaging, genomic analysis, and transcriptomic profiling to provide detailed insights into cellular behavior.

Nowadays, the noteworthy development is the integration of LOC technology with other emerging fields, such as AI and ML. AI and ML algorithms were employed to analyze the vast amounts of data generated by LOC devices, leading to improved accuracy, interpretation, and decision making in diagnostics and research.²⁴  Mencattini et al. propose combining microfluidics with ML approaches to enhance the diagnostic capabilities of LOC devices. They introduce a ML platform that utilizes deep learning to encode cell morphology beyond standard appearance. The platform is applied to a diagnostic test for blood diseases, specifically pyruvate kinase disease (PKD), achieving high accuracy in recognizing PKD from control red blood cells in both simulated and real experiments.²⁵  In recent years, the convergence of LOC technology with nanotechnology has further expanded its capabilities. Nanomaterials and nanostructures have been incorporated into chip designs, enabling enhanced sensitivity, specificity, and detection limits. This has facilitated the development of ultrasensitive biosensors, DNA sequencing platforms, and novel drug delivery systems.²⁶  For example, recently Cao et al. developed a robust LOC platform, for simultaneous quantification of multiple proteins using surface-enhanced Raman scattering (SERS) referred to as LOC‐SERS. The platform utilized antibody–DNA conjugates and Au nano-stars with Raman reporter molecules. With a gold-coated silicon nano-cone array as the capture substrate, the platform achieved ultrasensitive SERS detection with a limit of detection below pg mL⁻¹ levels. The platform showed excellent analytical performance and successfully analyzed protein biomarkers in a Parkinson’s disease mouse model.²⁷  Looking ahead to the near future, LOC technology is poised for even more transformative advancements. Continued integration of diverse analytical functionalities, improved automation, and standardization will contribute to its widespread adoption. LOC devices will become more user-friendly, enabling non-experts to perform complex analyses with ease. Furthermore, the integration of LOC technology with digital health platforms and telemedicine will revolutionize healthcare delivery and monitoring, empowering individuals to take charge of their health in a personalized manner.

Figure 1.1 shows a timeline that provides a general overview of the development of LOC devices and numerous other significant contributions that have been made by various research groups and companies.

LOC technology has offered many benefits in the field of the drug discovery process. Firstly, they provide high throughput and automation capabilities that make it possible to screen many compounds quickly and simultaneously using less material when compared with conventional approaches.²⁸  Therefore, there is a growing demand for low-cost technologies that reduce sample and reagent consumption, making microfluidics-based high-throughput drug screening platforms increasingly prominent. In recent years, significant progress has been made in the development of microfluidics-based drug screening components and systems.^29 ,30 

Secondly, in the field of drug delivery using LOC devices, drugs can be precisely delivered to particular parts/organs of the body using microfluidic channels, increasing their efficacy, and lowering the risk of side effects.³¹  These gadgets can be used to deliver medications to the affected organ continuously for a long time through regulated release. Additionally, LOC platforms can be engineered to replicate the physiological conditions of specific organs or tissues, facilitating the evaluation of precise drug delivery/targeting.³²  By emulating the microenvironment at the targeted drug delivery site, these devices enable more accurate evaluation and refinement of drug formulations. This ensures that drugs exhibit optimal efficacy in the intended area and can effectively navigate biological barriers or obstacles. Furthermore, the compact size of LOC devices provides further advantages for drug delivery. These miniature platforms necessitate smaller quantities of drugs, and they can be implanted within the body or utilized as portable devices. This portability enables flexible drug administration, either on demand or continuous, in various settings, including POC applications.^33 ,34 

Thirdly, LOC systems can be applied to disease modelling, enabling scientists to build simulations of disease processes and test potential treatments in a sterile setting. This can aid in understanding disease mechanisms and locating possible therapeutic targets.³⁵  For instance, LOC systems can be employed to recreate cancer tumor models, incorporating tumor cells, stromal cells, and extracellular matrix components.³⁶  This allows for the investigation of tumor growth, invasion, and response to different treatments. Through the observation of cancer cells interacting with their microenvironment and the testing of diverse drug candidates, researchers can deepen their understanding of cancer biology and develop more effective treatment strategies.³⁷ 

The versatility of LOC technology is shown in Figure 1.2 in the field of drug discovery for the management of health care. By cultivating pathogenic microorganisms, like bacteria or viruses, within microfluidic devices, researchers can analyze their behavior, mode of replication, and mechanism of interactions with host cells. This aids in comprehending disease transmission, host–pathogen interactions, and the development of novel antimicrobial approaches.^38–40  Moreover, LOC systems can be utilized to model genetic diseases, neurological disorders, cardiovascular conditions, and other complex diseases.^41 ,42  These platforms allow for the integration of multiple cell types, biochemical cues, and mechanical forces to recreate disease-specific microenvironments. By studying the behavior of cells and tissues in these disease models, researchers can gain insights into disease mechanisms, assess potential therapeutic interventions, and validate potential drug candidates before clinical trials.⁴³ 

LOC devices, being compact and portable, are highly suitable for POCT in various settings such as clinics, hospitals, and resource-limited areas. Their portability enables convenient and timely diagnosis of diseases in remote areas. These platforms offer a diverse range of diagnostic capabilities, including nucleic acid amplification, protein analysis, cell sorting, and immunoassays.⁴⁴  LOC devices can detect specific biomarkers associated with a wide array of diseases, such as infectious diseases, cancer, cardiovascular disorders, and metabolic conditions. This enables rapid and accurate detection of disease markers, supporting early diagnosis, disease monitoring, and personalized treatment strategies.^45 ,46 

By integrating multiple laboratory functions onto a single chip, LOC technology reduces the need for complex and time-consuming sample preparation steps. It enables efficient and automated analysis of samples, minimizing the risk of human error and enhancing result reproducibility.⁴⁷  Furthermore, these devices require smaller sample volumes, making them suitable for pediatric patients or situations where sample availability is limited. One significant advantage of LOC technology is its potential to reduce healthcare costs by streamlining the diagnostic process.⁴⁸  These devices provide rapid results, eliminating the need to send samples to centralized laboratories and wait for days to receive the outcomes. This not only improves patient care but also enables timely decision-making and optimizes the allocation of healthcare resources.⁴⁹  LOC devices have enormous potential for POC diagnostics for various diseases, including infectious diseases, cancer, and chronic diseases, and a summary of this is enlisted in Table 1.1.

LOC devices can be used for the rapid diagnosis of infectious diseases, such as COVID-19, influenza, and HIV. The identification of specific viral or bacterial antigens, antibodies, or nucleic acids, enables the detection of infections at an early stage. Rapid and accurate diagnosis of infectious diseases using LOC can help in timely treatment, reduce transmission, and prevent outbreaks. For instance, Zhu et al. developed a LOC device that combines DNA extraction, solid-phase PCR, and the genotyping method as shown in Figure 1.3(1). In this device the innovative design of pneumatic microvalves allowed for fluid mixing and reagent storage, reducing the chip size. The device successfully detected five high-risk HPV genotypes, with the capability of detecting around 50 copies of HPV virus per reaction in just one hour. This simple and cost-effective microdevice is highly useful for screening and monitoring HPV genotypes in POC molecular diagnostics.⁵⁰  In another study, Pennisi et al. developed a solution that combines host gene signatures with LOC technology for low-cost POC expression analysis to detect infectious diseases. They utilized portable and affordable LOC devices that incorporate ISFET and isothermal chemistries. The algorithm demonstrated a strong correlation with RT-qLAMP and achieved a remarkable classification accuracy of 100%. Their work aims to bring the advantages of microarray analysis to ISFET arrays, facilitating the detection of infectious diseases in remote areas.⁵¹  Likewise, using two sets of primers that target the open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2, Rodriguez-Mateos et al. developed an integrated on-chip platform that couples RNA extraction based on IFAST with RNA amplification and detection (Figure 1.3(2)) via colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP).⁵² 

LOC devices can also be used for the detection and monitoring of cancer biomarkers, such as circulating tumor cells, tumor-derived DNA, and microRNAs. These devices can enable the early detection of cancer, which can improve patient outcomes and reduce healthcare costs. Alexandrou et al. conducted a study showcasing the effectiveness of an integrated LOC system that employs ISFET technology and variant-specific isothermal amplification. The system successfully detected and differentiated between wild-type (WT) and mutant (MT) copies of the ESR1 gene. The optimized LOC assays demonstrated fast amplification (within 25 minutes) and high sensitivity (detecting as few as 1000 DNA copies per reaction). This compact and cost-efficient LOC platform has promising applications in POCT for breast cancer, facilitating patient stratification and metastatic disease monitoring through liquid biopsies.⁵³  Additionally, LOC devices can be used to monitor the effectiveness of cancer treatments and track disease progression.

LOC devices can be used for the monitoring and management of chronic diseases, such as diabetes, cardiovascular diseases, and respiratory diseases. These devices can measure various biomarkers, such as glucose, cholesterol, and inflammatory markers, enabling patients to monitor their health status in real time and make informed decisions about their treatment. For instance, Vutthikraivit et al. (2021) created and validated a biosensor using LFA to detect and measure albumin in the urine of patients with CKD (shown in Figure 1.3(3)). Monoclonal antibodies against human serum albumin were selected and used in the biosensor. The performance of this LOC system was compared to other POCTs and gold-standard methods for albumin detection, using urine samples from CKD and diabetes patients. The LFA-based biosensor exhibited a sensitivity of 86%, specificity of 94%, and positive predictive value of 96%.⁵⁴ 

The role of LOC devices in POC diagnostics is significant, particularly in resource-limited settings, where access to laboratory facilities and trained personnel is limited. LOC devices can be used in a variety of settings, including clinics, hospitals, and even in the field, enabling the rapid diagnosis and treatment of various diseases.⁵⁵  In summary, LOC devices have the potential to transform POC diagnostics by enabling rapid, accurate, and cost-effective diagnosis of various diseases. These devices have numerous applications in infectious diseases, cancer, and chronic diseases and can improve patient outcomes and reduce healthcare costs.

LOC devices have a critical role in therapeutic designing and discovery, providing faster and more efficient methods for screening and identifying potential drug candidates briefly described in Table 1.2. The development of new drugs is a complex and time-consuming process, with high costs and low success rates. LOC devices can improve the drug discovery process by reducing the time and costs involved in identifying new drug candidates.

LOC devices can be used for HTS of potential drug candidates, enabling the rapid screening of thousands of compounds in a short time. These devices can automate the drug screening process, allowing researchers to test multiple drug candidates simultaneously, reducing time and costs. Automation plays a crucial role in HTS, as it leverages robotic systems and automated processes to handle and test thousands to millions of compounds in a short period. The development of robust and miniaturized assays is essential for HTS, allowing for the measurement of specific biological activities or interactions between compounds and targets.⁵⁶  Diverse compound libraries, containing small molecules, natural products, or other bioactive substances, are employed in HTS to provide a wide range of potential hits. Data analysis is a critical aspect of HTS, involving advanced computational methods and statistical analysis to interpret the large amounts of data generated during screening.⁵⁷  Identified hits are further validated, optimized, and subjected to follow-up experiments, hit confirmation assays, and structure–activity relationship studies to ensure their specificity and efficacy. HTS finds applications in target identification, lead compound identification, optimization, and is also used in chemical biology, functional genomics, and proteomics.⁵⁸  Technological advancements, such as LOC devices, miniaturization, and high-content imaging systems, have significantly improved the efficiency, speed, and accuracy of HTS. Overall, HTS is a powerful tool in therapeutic design and drug discovery, facilitating the identification of potential candidates for further development.

LOC devices can create microenvironments to mimic in vivo conditions, enabling the testing of drug candidates under realistic physiological conditions. Microfluidic platforms can also enable the study of drug interactions with cells, tissues, and organs, providing valuable insights into drug efficacy and safety. One notable application of microfluidics is in HTS, where it has enabled the miniaturization and automation of various screening processes.^59 ,60  This includes cell-based assays and enzyme kinetics, enabling the rapid and parallel analysis of multiple samples. This advancement reduces reagent consumption, increases screening efficiency, and accelerates the screening of large compound libraries.⁶¹  Microfluidics has also facilitated the development of organ-on-a-chip models, which replicate the physiological environment of specific tissues or organs. These models provide a more realistic and controlled setting for studying drug responses, toxicity, and disease mechanisms.⁶¹  Microfluidic systems allow for precise control of overflow rates, shear stress, and nutrient gradients, mimicking conditions found in the human body. In the realm of drug delivery systems, microfluidic devices offer precise control over drug release kinetics, aiding in the design and optimization of targeted and sustained-release formulations.^59 ,61  Additionally, microfluidics can be utilized to study drug transport across barriers, such as the blood–brain barrier, enhancing our understanding of drug delivery challenges. Microfluidics has also greatly benefited LOC diagnostics by integrating sample preparation, amplification, and detection methods in a miniaturized format.⁶²  This enables the development of portable and POC diagnostic platforms that provide rapid and sensitive detection of biomarkers for disease diagnosis and monitoring. Furthermore, microfluidics plays a crucial role in single-cell analysis, allowing for the isolation, manipulation, and analysis of individual cells.⁶³  This technology facilitates the study of cellular heterogeneity, drug responses at the single-cell level, and the identification of rare cell populations. Microfluidic devices enable high-resolution analysis of cellular characteristics, such as gene expression or protein levels, leading to advancements in our understanding of disease mechanisms and therapeutic responses.

LOC devices can be used in personalized medicine to develop drugs tailored to individual patients LOC technologies enable the high-throughput, fast screening, and characterization of prospective drug candidates in therapeutic designing and discovery. These tools make it possible to integrate several screening procedures, such as sample preparation, target identification, and efficacy testing, as well as the exact control of fluidic conditions.⁶⁴  LOC systems can process and analyze patient samples quickly because of their compact size and automation capabilities, making it easier to find personalized treatment alternatives.⁶⁵  In addition, LOC technologies enable the creation of patient-specific tests and diagnoses. These devices may identify and analyze biomarkers or genetic changes linked with a patient’s disease or treatment response by merging technologies such as microfluidics, biosensors, and nucleic acid amplification.^64 ,66  This data may be used to influence treatment decisions, allowing for more personalized therapeutic regimes.

LOC devices can also be used for drug delivery, enabling precise control over the dosage and timing of drug delivery. These devices can target specific tissues and organs, reducing the risk of adverse effects and improving drug efficacy.⁶⁷  LOC technology holds great promise for revolutionizing drug delivery methods. With its ability to integrate multiple functions onto a small chip, LOC devices offer precise and controlled drug delivery systems.⁶⁸  One key application is the use of microfluidics, where microchannels and chambers allow for the accurate loading of drugs onto the chip, ensuring controlled dosing and minimizing the risk of improper drug administration.⁶⁹  Moreover, LOC devices can be designed to enable targeted drug delivery by mimicking specific physiological environments, facilitating the precise delivery of drugs to target sites in the body while minimizing exposure to healthy tissues. This approach enhances therapeutic efficacy and reduces side effects.⁷⁰  On-chip drug release systems further enhance control by incorporating micro-reservoirs or microvalves, enabling programmable drug delivery profiles tailored to individual treatment requirements.⁷¹  Real-time monitoring and feedback mechanisms, such as sensors and detectors integrated into the chip, allow for continuous monitoring of drug release, drug concentration, and physiological parameters at the site of drug delivery.⁷²  This monitoring capability enables adaptive and personalized drug delivery, where treatment can be adjusted based on patient responses, optimizing therapeutic outcomes. Additionally, LOC technology enables the integration of multiple drugs or therapeutic agents on a single chip, facilitating combination therapy approaches that enhance efficacy and mitigate drug resistance.⁷³  The miniaturized and portable nature of LOC devices also makes them suitable for POC applications. They can be utilized in clinics, hospitals, or even at home, enabling on-site drug delivery and personalized medicine, ultimately reducing the need for frequent hospital visits.⁷⁴  These advancements in LOC technology present exciting opportunities for more precise, targeted, and patient-centric drug delivery methods.

In addition to these applications, LOC devices can also improve the drug discovery process by reducing the need for animal testing, increasing the reproducibility of results, and enabling the development of drugs for rare diseases. Taken together, LOC devices have a critical role in therapeutic design and discovery, providing faster, more efficient, and cost-effective methods for screening and identifying potential drug candidates. These devices have numerous applications and are expected to drive innovation and progress in the field of drug discovery.

LOC devices can be used for HTS of potential drug candidates. These devices allow researchers to test multiple compounds simultaneously, reducing the time and cost associated with traditional drug screening methods. Microfluidics, as an emerging application of LOC technology, has garnered significant interest in the field of pharmaceutical science.⁷⁵  The growing demand for LOC technologies in pharmaceutical research has driven the development of Pharm-LOC concept. This innovative approach encompasses chip-based platforms that cater to the entire range of pharmacy applications, spanning from drug discovery to post-marketing product management.⁷⁶  Pharm-LOC integrates various aspects of chip-based principles, techniques, and devices, enabling pharmaceutical analysis, pharmacological and toxicological testing, as well as pharmaceutical production. In essence, it leverages microfluidics to advance and streamline multiple facets of pharmaceutical science, revolutionizing the way drugs are developed, tested, and manufactured.⁷⁷  For instance, in response to the urgent need for rapid drug development to combat the rapidly spreading and evolving coronavirus, a key challenge lies in effectively constructing the cell membrane at the molecular level. A label-free SLB-based LOC biosensor for the effective screening of inhibitory medicines was proposed by Zhou et al. as a solution to this problem. The biosensor features an expanded gate electrode that is functionalized with a SLB that contains an angiotensin-converting enzyme-2 (ACE2) receptor. The schematic process of developed LOC is shown in Figure 1.4(1). This integrated system simulates the in vitro cell membrane microenvironment while minimizing interference by translating target–receptor interactions into real-time charge signals.⁷⁸  Another researcher, Zeid et al. developed a precise and sensitive LOC electrophoretic method coupled with light-emitting diode-induced fluorescence (LED-IF) detection for the analysis of three antiepileptic drugs: vigabatrin, pregabalin, and gabapentin in pharmaceutical formulations. The method achieved rapid separation of the drug mixture with good resolution and high theoretical plate numbers. By employing stacking, the sensitivity of the method was significantly enhanced, reaching a detection limit below 0.6 ng mL⁻¹ in the concentration range of 2.0–200.0 ng mL⁻¹. The developed method was successfully validated for the analysis of the studied drugs in pharmaceutical formulations.⁷⁹  High-content screening (HCS) is a crucial tool in biological applications and drug development. Huang et al. have developed microfluidic chips that allow simultaneous screening of 10 types of drugs, each at 5 different concentrations. Their imaging system captures videos at a 30 Hz rate, covering a centimeter field-of-view with a resolution of 0.8 μm. By utilizing this HCS system, they successfully assayed the effects of 12 small molecules on the Ca²⁺ ion signal of cardiomyocytes. This novel HCS paradigm and state-of-the-art platform present an advanced alternative to traditional well-plate-based methods.⁸⁰  These devices allow researchers to test multiple compounds simultaneously, reducing the time and cost associated with traditional drug screening methods. Some examples of drug discovery processes using LOC devices have been enlisted in Table 1.2.

LOC devices can be used for precise drug delivery, enabling drugs to be delivered to specific areas of the body. Conventional drug delivery methods administer drugs through various routes such as oral, transdermal, inhalation, or injection. However, these methods can be ineffective for certain drugs susceptible to enzymatic degradation.⁸¹  Recent advancements in micro- and nanotechnologies offer targeted and shortened delivery pathways. The integration of drug delivery components into a single chip, known as LOC, allows for miniaturization and precise control.⁸²  LOC technology, based on microfluidics, impacts drug delivery from drug carrier synthesis to screening and the delivery system itself. Microbubbles have been utilized in ultrasound-assisted drug delivery to target solid tumors in vivo through blood vessels.⁸³  Park et al. developed a microfluidic microvessels-on-a-chip system to visualize the delivery of drugs using microbubbles and ultrasound shown in Figure 1.4(2). Minimal cellular damage was observed for both microbubbles and untargeted doxorubicin-encapsulating liposomes perfused through the chip’s microvessels. Additionally, the microvessels-on-a-chip system proved valuable as a screening platform for optimizing drug dosage and targeting ligands, and drugs.⁸⁴  Recently, researchers have developed a method to construct hierarchical and perfusable vessel networks for on-chip tissue models. Zhou et al. induced spontaneous anastomosis between endothelialized lumens and self-assembled capillary networks, resulting in a perfusable network with vessels at different scales. This approach enabled the creation of an in vivo-like hierarchical vessel-supported tumor model, which was used for anticancer drug testing. Combining computational fluid dynamics simulation and a tumor growth mathematical model, the vessel perfusability effect on tumor growth rate was predicted. The hierarchical vessel-supported tumor exhibited a higher growth rate and drug delivery efficiency compared to the tumor model without capillary vessels.⁸⁵  Nanomedicine has improved drug delivery, but cellular uptake in vivo remains limited. Traditional 2D culture models do not accurately represent in vivo conditions. Zhuang et al. developed a MTC-chip to mimic solid tumors and fluid transport, allowing a better study of nanoparticle penetration. The chip was used to evaluate cellular uptake of mesoporous silica particles (MSNs). Continuous administration resulted in greater MSNs penetration compared to transient administration at the same dose. The size effect on cellular uptake was less significant than previous in vitro studies. Pre-treatment with hyaluronidase (HAase) improved the tumor penetration of large-size MSNs.⁸⁶  This can improve the efficacy of the drugs and reduce their side effects. These devices can also be used for the controlled release of drugs, enabling sustained drug delivery over an extended period.

LOC devices can be used for disease modelling, allowing researchers to create models of disease processes and test potential treatments in a controlled environment. This can help researchers understand the mechanisms of diseases and identify potential drug targets. LOC technology-based modelling of diseases intends to replicate and investigate the intricate physiological circumstances linked to various diseases.^87 ,88  To imitate disease processes, LOC offers a regulated and microscale environment that permits the integration of many components, including cells, tissues, and fluidic systems. Researchers can develop in vitro models that closely reflect the pathophysiology of diseases by embedding disease-specific cells or tissues onto microfluidic devices.⁸⁹  Micro-physiological systems, such as micro engineered tissues and organoids, have shown great potential in replicating tissue histogenesis and biological functions for drug testing and disease modelling. However, these systems lack certain crucial features, including tissue interfaces, vascular perfusion, interstitial flow, immune cell circulation, and organ-specific mechanical cues. On the other hand, microfluidic organ chips offer the ability to incorporate these functions, making them more suitable for accurately assessing drug disposition, efficacy, and toxicity within the human body.⁹⁰  Pediaditakis et al. utilized Organs-on-Chips technology to create a human Brain-Chip representing the substantia nigra region of the brain affected by Parkinson’s disease. This model shown in Figure 1.4(3) incorporated various cell types, including dopaminergic neurons, astrocytes, microglia, pericytes, and brain endothelial cells, cultured under fluid flow conditions. By inducing αSyn fibril formation, the model successfully replicated key features of Parkinson’s disease, including the accumulation of phosphorylated αSyn, mitochondrial dysfunction, neuroinflammation, and compromised barrier function. This Brain-Chip provides a valuable tool for studying cell–cell interactions in synucleinopathies and testing novel therapeutics.⁹¹ 

Another research group, Hachey et al., has validated a microphysiological system (MPS) platform for studying CRC, the second leading cause of cancer-related deaths. Their research demonstrates that this platform more accurately models gene expression, tumor heterogeneity, and treatment responses compared to standard drug screening methods such as two-dimensional monolayer culture and three-dimensional spheroids.⁹²  This advancement in in vitro models could improve drug screening and disease modelling for CRC. Recently, a human organ-on-a-chip microfluidic device was developed by Goyal et al. to mimic ectopic lymphoid follicle formation. Primary human B- and T-lymphocytes cultured in a 3D extracellular matrix gel within the device autonomously formed lymphoid follicles. The model demonstrated potential for studying lymphoid follicle development and evaluating vaccines.⁹³  The effectiveness of drugs or therapies can be evaluated using these models, and they can also be used to find possible biomarkers for use in diagnosis and therapy monitoring.

Microfluidic chip development focuses on the manipulation and control of fluids at a micro/sub-micron scale. Initially, these chips were developed as alternatives to traditional laboratory analysis methods by creating “μ-TAS” or “LOC” models incorporating micro chromatography and capillary electrophoresis.⁹⁴  To simulate the harmful effects of drug candidates on specific cells, tissues, or organs in the human body, organ-on-a-chip models have been developed. Individual organs such as the liver, kidney, heart, and nerves, as well as multi-organs-on-chips, are replicated in these models. As the technology advanced and gained wider acceptance, microfluidic chips found applications in the fields of cell biology and cell analysis.^95 ,96  This led to the development of on-chip cell culture techniques, the simulation and replication of in vivo microenvironments, single-cell analysis chips, and more advanced models like “organ-on-a-chip” systems. Preclinical toxicity testing is critical for identifying and treating drug toxicity, which is a major cause of post-market drug withdrawal.⁹⁷  However, due to species differences, the application of animal model results to humans is limited. Various in vitro models for toxicity screening have been created to improve the accuracy of preclinical toxicity testing, with organ-on-a-chip emerging as a viable option. The liver is a crucial organ susceptible to drug toxicity, and drug-induced liver injury (DILI) can lead to the withdrawal of drugs from the market as well as acute and chronic liver diseases.^95 ,98  Detecting drug hepatotoxicity is a critical aspect of drug development, and liver-on-a-chip technology has emerged as a cutting-edge technique in this regard. Perfusion culture technology is commonly used to culture hepatic cells in liver chips. Recently, researchers developed a hepatic sinusoidal chip device with four transformed cell lines, employing a peristaltic pump for perfusion. This method replicated the physiological characteristics of hepatic sinusoids, improving the sensitivity of hepatocellular carcinoma (HepG2) cell toxicity testing and facilitating drug–drug interaction studies.⁹⁸  Apart from peristaltic perfusion, other effective methods such as gravity-driven, paper-based siphon, and CO₂ gas-driven perfusion have also been applied in liver chips. Yu et al. constructed a perfusion-incubator-liver-chip (PIC) that integrated air bubbles and temperature processing capabilities. The flow in the chip was driven using CO₂ pressure, enabling pH control and maintaining liver cell viability for up to 14 days with sustained functionality. Similarly, the kidney-on-a-chip model has been used to investigate nephrotoxicity since it allows for cell co-culture, simulation of the in vivo milieu, and in vitro biomarker profiling.⁹⁹  Sakolish’s group employed immortal cell lines to imitate glomerulus and proximal tubule activity. The platform shown in Figure 1.5(1) included a glomerular filter containing human umbilical vein endothelial cells (HUVECs) to give a more realistic “primary urine” inside the device, in addition to providing a shear-stressed culture environment for HK-2 cells.¹⁰⁰  Later, the researchers employed a similar apparatus to assess the DIN by cisplatin and cyclosporine. According to the findings, proximal tubular human kidney-2 (HK-2) cells cultivated in this microfluidic system are more sensitive. Studies revealed that fluid shear stress could increase the retention of more mature phenotypes in proximal tubular epithelial cells and the development of a glomerulus-on-a-chip model.^99 ,101 

LOC devices can be used for POC diagnostics, enabling the rapid and accurate diagnosis of diseases in a clinical setting. These devices can be used to detect a range of diseases, including infectious diseases, cancer, and genetic disorders. LOC devices are advanced microfluidic devices that integrate and automate various functions, including sample preparation, reagent manipulation, detection, and result analysis, within a single platform.¹⁰²  These devices have greatly contributed to the miniaturization, portability, integration, and automation of multiple assay functions in POC devices. In comparison to traditional detection methods, highly integrated LOC devices offer numerous advantages such as rapid detection, ease of multiplexing and integration, minimal reagent consumption, cost-effectiveness, high accuracy, and portability.^102 ,103  As an example, for clinical POC HIV diagnoses employing an RT-PCR-based chemiluminescence assay, Lee et al. created a revolutionary disposable polymer-based LOC device. On-chip RT-PCR and a portable analyzer were combined flawlessly by the device. The reverse transcription process was controlled by a non-contact infrared temperature control system in the analyzer, and the RT-PCR LOC was detected and monitored on-chip using an optical detection system. In less than an hour, the created polymer LOC device proved its capacity to detect HIV POC while reducing the chance of cross-contamination.¹⁰⁴  Likewise, Qin et al. developed a simplified LOC device for automated POC detection of the Ebola virus shown in Figure 1.5(2).¹⁰⁵  Their device combined a CRISPR microfluidic chip with a custom fluorometer, enabling rapid and sensitive detection using a small sample volume of 10 μL. The device facilitated automated mixing, hybridization, and measurement of Cas13a cleavage products, achieving detection of total Ebola RNA in just 5 minutes without solid-phase extraction. The device demonstrated a low detection limit of approximately 1 × 10⁻³ ng mL⁻¹. LOC-POCT devices for cancer treatment also garnered a great deal of attention from researchers. Wormald et al. demonstrated the efficacy of a handheld LOC device in detecting cervical cancer from biopsy samples. The device employed ISFET sensors and LAMP assays to amplify HPV DNA and hTERT mRNA, achieving high sensitivity and potential for large-scale screening in resource-limited settings.¹⁰⁶ 

The market potential for LOC devices is significant, backed by market predictions, and driven by several reasons. With a compound yearly growth rate of 22.6% from 2020 to 2027, the global market for microfluidic devices, which includes LOC devices, is anticipated to increase to $13.24 billion by the end of that period. The influence of LOC devices on drug development is a major factor influencing market potential. These technologies are appealing to pharmaceutical and biotech businesses because they offer the potential to speed up the drug discovery process and lower development expenses. The use of LOC devices in POC diagnostics is another aspect. These gadgets can enhance patient outcomes and save healthcare costs by providing quick and accurate illness identification in a clinical environment. The potential market for LOC devices is also being boosted by the idea of personalized treatment. These devices can boost efficacy and minimize negative effects by customizing therapies for each patient, which has major advantages for both patients and healthcare professionals. Additionally, LOC devices are useful in several scientific disciplines, such as biology, chemistry, and materials science. Their adaptability might be advantageous to academic and government research institutes, increasing business potential in various fields. However, several issues affect the commercial potential for LOC devices, including legal restrictions, opposition from established and new technologies, and financial constraints. For LOC devices to reach their full market potential, it will be essential to overcome these obstacles.

For more than three decades, LOC technologies, also known as μ-TAS, have had a significant impact by integrating analysis methods into devices and systems. These technologies allow for the analysis of real samples on micro-engineered chips and necessitate a highly multidisciplinary approach, as shown in Figure 1.6, that combines disciplines such as chemistry, engineering, computational science, biological physics, life sciences, genetics, molecular biology, environmental, clinical, and veterinary sciences.^98 ,107 

The integration of diverse subject areas and technologies has already demonstrated remarkable examples of performing analysis outside of traditional laboratory settings. The compact size of LOC devices allows for reduced sample volumes, rapid mass transfer over shorter distances, and the utilization of nanoscale materials with unique properties in sensors.¹⁰⁸  Commercially available LOC sensors offer sample-to-answer solutions, including finger-stick and continuous glucose monitoring, as well as paper-based microfluidic lateral flow technologies commonly used in pregnancy testing. These immunoassay technologies have also found extensive applications in medical diagnostics, such as testing for endemic infectious diseases like malaria, and more recently, facilitating community-based SARS-CoV-2 detection during the ongoing pandemic.^109 ,110 

LOC technologies utilize micro-engineered devices to perform sample preparation, analyte separation, and detection in miniaturized formats, enabling testing outside of traditional laboratories. Despite their advantages, challenges persist. For instance, the need to shift diagnostic tests, including sensitive molecular testing, from specialized labs to community settings is evident, as seen in the COVID-19 pandemic.¹¹¹  Lateral flow tests on paper microfluidics offer diagnostics at home or work, but more sensitive molecular testing on low-cost, autonomous LOC systems is desired. These technologies are crucial for global infectious disease management and the elimination of endemic diseases like schistosomiasis, malaria, and hepatitis.^111 ,112  Future challenges include developing cost-effective, disposable platforms for “sample-to-answer” analysis. Improved molecular diagnostics are essential to identify disease reservoirs in remote and underserved communities, detecting asymptomatic and pre-symptomatic individuals. Key requirements involve sensitive tests utilizing biomarker amplification, such as PCR or isothermal assays. For RNA viruses like SARS-CoV-2, Ebola, and hepatitis, the sample preparation step must address the additional challenge of reverse transcription to convert RNA into DNA before amplification.¹¹³ 

In recent years, LOC technologies have incorporated AI and ML through expert systems and deep learning algorithms. These AI-driven systems provide decision support for clinical and environmental analysis, particularly in remote or low-resource settings where laboratory facilities are lacking. Mobile phones equipped with data collection capabilities and deep learning algorithms enable diagnostic decision support, shaping treatment pathways in such settings.¹¹⁴  The integration of LOC systems with central medical databases and secure digital health infrastructure remains a challenge that requires a multidisciplinary approach. Intelligent microfluidics is an emerging field that utilizes ML for monitoring and controlling microfluidic systems. This development opens opportunities for accelerating chemical exploration and synthesis, leading to advancements in drug discovery, materials science, and digital chemistry.¹¹⁵ 

The convergence of microfluidic chip fabrication and tissue engineering has given rise to organs-on-a-chip and organoid research. The integration of machine intelligence into artificial biological organs presents a new challenge in predicting in vitro drug efficacy and toxicity, thereby aiding the development of safer and more effective pharmaceuticals.¹¹⁶  Altogether, the incorporation of AI and ML into LOC technologies holds promise for enhanced decision support, high-throughput discovery, and advanced organ modelling, while posing challenges related to data integration, security, and the ethical implications of AI-driven diagnostics and research.¹¹⁷ 

LOC technology faces critical challenges in meeting global sustainability and net-zero targets. This involves developing smart analytical systems that can detect environmental changes and addressing the use of single-use plastics and low-power, recyclable microsystems technologies.¹¹⁸  LOC devices offer the ability to monitor pollutants and toxins in the environment, providing simple sample-to-answer solutions for scientists to assess environmental changes. Future advances may include systems capable of measuring the effects of warming season microflora and microfauna. These systems will utilize deep learning and data logging to predict trends and collect continuous environmental data. In terms of sustainability, most LOC devices are currently single-use and contribute to growing amounts of medical waste. Exploring biodegradable materials, such as cellulose or paper derived from natural sources, may replace non-renewable plastic materials.^118 ,119  Adopting a circular economy approach and considering the carbon footprint of LOC devices will be essential for a more sustainable future. Expert systems, particularly in digital health, can reduce the environmental footprint of measurement systems by providing crucial information at the point of need, minimizing transport costs and infrastructure burdens.¹²⁰  Local manufacturing, as exemplified by initiatives like Diatropix in Senegal, can contribute to net-zero goals by producing diagnostic devices close to where they are required.¹²¹ 

Prominent figures in the field of LOC will be encouraged to share their perspectives on how technological advancements will shape society in the coming years. This includes considering the implications of new sustainable manufacturing technologies that aim to reduce production costs and enhance product reliability without compromising the environment.¹²²  The evolution of manufacturing methods will involve optimizing and streamlining existing micro- and nano-lithographic techniques, as well as integrating unconventional approaches like hybrid LOC manufacturing using additive manufacturing methods such as 3D printing and nanoscale self-assembly.¹²³  Additionally, molding, embossing, and reel-to-reel processing techniques have been adapted to create hybrid devices at both micro- and macro scales. These innovative manufacturing methods pave the way for more efficient and eco-friendly production of LOC devices.¹²⁴ 

LOC devices have a bright future in drug discovery due to their numerous advantages over traditional methods. The future of LOC technologies seems bright as due to advances in miniaturization, integration, and automation in the field of personalized medicine (Figure 1.7). These advancements enable more efficient and high-throughput sample analysis in a variety of disciplines, including healthcare, environmental monitoring, and research. Enhancing sensitivity and detection limits, as well as pushing the frontiers of analytical performance, are key areas of attention for future development of LOC systems.¹³  A major emphasis is also placed on improving sample preparation processes to ensure precise and trustworthy findings. Furthermore, a seamless connection with digital health systems, allowing for real-time data gathering, analysis, and decision support, is a need. To reduce the environmental effect of LOC technologies and ensure their wider adoption, sustainable manufacturing practices are being sought. As a result of these improvements, LOC technologies are set to revolutionize diagnostics, monitoring, and scientific research, opening up new avenues for precision medicine, personalized healthcare, and speedy on-site POCT.

LOC technologies have the potential to completely change the way drugs are discovered by offering quicker, more effective, and more affordable ways to screen and develop new drugs. Compared to conventional techniques, LOC devices have many advantages, such as miniaturization, HTS, automation, and integration. LOC device development and adoption, however, are obstructed due to several challenges such as complexity, standardization, integration, materials, and cost are some major concerns. To fully realize the potential of LOC devices in drug discovery, these difficulties must be overcome. Despite these difficulties, there is a sizable market for LOC devices due to the rising need for microfluidic devices in research, POC diagnostics, personalized medicine, and drug discovery. Regulatory constraints, rivalry, and financing availability all have an impact on the commercial potential for LOC devices, which could affect their wide usage and success. Overall, LOC devices have a bright future in the field of drug discovery, and their continuing advancement and fusion with other technologies will spur innovation and advancement in the field of health care.

The authors would like to thank the director of CSIR-AMPRI for his guidance and inspiration in this project. The fellowship offered by DST (DST/WOS-B/HN-4/2021) is duly acknowledged by Arpana Parihar.